Colorectal cancer (CRC) is the 4th most common cancer and the 2nd leading cause of cancer?related deaths in the United States. The American Cancer Society estimates that 134,490 people will be diagnosed and 49,190 will die from CRC in 2016. Although biologic agents have received much attention, the first-line chemotherapy for treatment of metastatic CRC remains based on a cytotoxic combination chemotherapy backbone of either FOLFIRI (folinic acid+5-FU+irinotecan) or FOLFOX (folinic acid+5-FU+oxilaplatin). It is noteworthy that only 18-25% of CRC patients respond favorably to irinotecan. Although the FDA-approved irinotecan has been used in clinic for 20 years, there is still no diagnostic biomarker to help oncologists decide which of the two regimens is more likely to be effective for a given CRC patient. Our studies suggest that the CULLIN 4B (CUL4B) ubiquitin ligase is a promising predictive biomarker, as well as a therapeutic target for tumor response to irinotecan. The CUL4B gene has been found amplified or overexpressed in a wide range of solid tumors, including colorectal, breast, lung, ovarian, and prostate cancers. Upon treatment with the camptothecin family of chemotherapy drugs, CUL4B, but not its paralog CUL4A, targets topoisomerase I (Top1) for degradation in cancer cells. As a result, CRCs with high levels of CUL4B expression induce excessive destruction of Top1, effectively attenuating the cytotoxicity of this chemotherapy drug. We hypothesize that excessive Top1 degradation by CUL4B is a major mechanism by which CRCs become refractory to irinotecan. Importantly, we showed that inactivation of CUL4B, but not CUL4A, effectively sensitized irinotecan-resistant CUL4Bhigh tumors to cytotoxic killing by Top1-directed chemotherapy drugs. This data suggest that CUL4B is an attractive target for intervention, potentially leading to sensitization of the 75-82% CRC patients who were previously resistant to treatment with a Top1-directed chemotherapeutic agent (e.g. FOLFIRI regimen). In three specific aims, we will (1) assess the value of CUL4B as a biomarker for treatment decisions of CRC using our unique collection of 572 well annotated CRC patient samples treated with irinotecan or FOLFIRI; (2) further develop and validate potent small molecule CUL4B inhibitors we identified via high throughput screening to increase their potency and pharmacological properties; (3) examine our lead CUL4B inhibitors that synergize with irinotecan to enhance its tumoricidal activity in vivo using CRC cell line xenografts, genetic WNT/APC-induced intestinal and colon adenomas, and patient-derived tumor xenograft (PDTX) models of CRC. We are uniquely prepared for both Laboratory-to-Clinic studies to develop a predictive biomarker for irinotecan-based chemotherapy that is immediately applicable for informing decision- making of CRC management in the clinic, and Clinic-to-Laboratory studies aimed at validating CUL4B as a feasible drug target in pre-clinical CRC models, and developing a new therapeutic agent to render CUL4Bhigh CRC patients, which represent approximately 75% of all CRCs, sensitive to irinotecan-based therapy.